Browsing by Author "Hohmann, S."
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- Existence of a tightly regulated water channel in saccharomyces cerevisiaePublication . Meyrial, V.; Laizé, Vincent; Gobin, R.; Ripoche, P.; Hohmann, S.; Tacnet, F.The Saccharomyces cerevisiae strain Σ1278b possesses two putative aquaporins, Aqy1-1p and Aqy2-1p. Previous work demonstrated that Aqy1-1p functions as a water channel in Xenopus oocyte. However, no function could be attributed to Aqy2-1p in this system. Specific antibodies were used to follow the expression of Aqy1-1p and Aqy2-1p in the yeast. Aqy1-1p was never detected whatever the growth phase and culture conditions tested. In contrast, Aqy2-1p was detected only during the exponential growth phase in rich medium containing glucose. Aqy2-1p expression was repressed by hyper-osmotic culture conditions. Both immunocytochemistry and biochemical subcellular fractionation demonstrated that Aqy2-1p is located on the endoplasmic reticulum (ER) as well as on the plasma membrane. In microsomal vesicles enriched in ER, a water channel activity due to Aqy2-1p was detected by stopped-flow analysis. Our results show that the expression of aquaporins is tightly controlled. The physiological relevance of aquaporin-mediated water transport in yeast is discussed.
- Molecular and functional study of AQY1 from Saccharomyces cerevisiae: role of the C-terminal domainPublication . Laizé, Vincent; Gobin, R.; Rousselet, G.; Badier, C.; Hohmann, S.; Ripoche, P.; Tacnet, F.The yeast YPR192w gene, which encodes a protein (Aqy1p) with strong homology to aquaporins (AQPs), was cloned from nine S. cerevisiae strains. The osmotic water permeability coefficient (Pf) of X. laevis oocytes expressing the gene cloned from the S1278b strain (AQY1-1) was 5.7 times higher than the Pf of oocytes expressing the gene cloned from other strains (AQY1-2). Aqy1-1p, initially cloned without its C-terminus (Aqy1-1DCp), mediated an ;3 times higher water permeability than the full-length protein. This corresponds to a 3-fold higher protein density in the oocyte plasma membrane, as shown by freeze-fracture electron microscopy. Pf measurements in yeast spheroplasts confirmed the presence of functional water channels in S1278b and a pharmacological study indicated that this strain contains at least a second functional aquaporin.
- Polymorphism of Saccharomyces cerevisiae aquaporinsPublication . Laizé, Vincent; Tacnet, F.; Ripoche, P.; Hohmann, S.Aquaporin water channels facilitate the transmembrane diffusion of water and higher organisms possess a large number of isoforms. The genome of the yeast Saccharomyce cerevisiae contains two highly similar aquaporin genes, AQY1 and AQY2. AQY1 has been shown to encode a functional water channel but only in certain laboratory strains. Here we show that the AQY2 gene is interrupted by an 11 bp deletion in 23 of the 27 laboratory strains tested, with the exception of strains from the S1278b background, which also exhibit a functional Aqy1p. However, although the AQY2 gene from S1278b is highly homologous to functional aquaporins, we did not observe Aqy2p mediated water transport in Xenopus oocytes. A survey of 52 yeast strains revealed that all industrial and wild yeasts carry the allele encoding a functional Aqy1p, while none of these strains appear to have a functional Aqy2p. We conclude that natural and industrial conditions provide selective pressure to maintain AQY1 but apparently not AQY2.
- Ser3p (Yer081wp) and Ser33p (Yil074cp) are phosphoglycerate dehydrogenases in Saccharomyces cerevisiaePublication . Albers, E.; Laizé, Vincent; Blomberg, A.; Hohmann, S.; Gustafsson, L.Two genes YER081W and YIL074C, renamed SER3 and SER33, respectively, which encode phosphoglycerate dehydrogenases in Saccharomyces cerevisiae were identified. These dehydrogenases catalyze the first reaction of serine and glycine biosynthesis from the glycolytic metabolite 3-phosphoglycerate. Unlike either single mutant, the ser3Delta ser33Delta double mutant lacks detectable phosphoglycerate dehydrogenase activity and is auxotrophic for serine or glycine for growth on glucose media. However, the requirement for the SER-dependent "phosphoglycerate pathway" is conditional since the "glyoxylate" route of serine/glycine biosynthesis is glucose-repressed. Thus, in cells grown on ethanol both expression and activity of all SER-encoded proteins are low, including the remaining enzymes of the phosphoglycerate pathway, Ser1p and Ser2p. Moreover the available nitrogen source regulates the expression of SER genes. However, for only SER33, and not SER3, expression was regulated in relation to the available nitrogen source in a coordinated fashion with SER1 and SER2. Based on these mRNA data together with data on enzyme activities, Ser33p is likely to be the main isoenzyme of the phosphoglycerate pathway during growth on glucose. Moreover, since phosphoglycerate dehydrogenase activity requires NAD(+) as cofactor, deletion of SER3 and SER33 markedly affected redox metabolism as shown by substrate and product analysis.
- Transposon mutagenesis reveals novel loci affecting tolerance to salt stress and growth at low temperaturePublication . Ferreira, MC De Jesus; Bao, X.; Laizé, Vincent; Hohmann, S.Using transposon mutagenesis in the haploid Saccharomyces cerevisiae strain W303-1A we have identified genes required for growth in high salt medium, survival of a hypo-osmotic shock and growth at 15 degrees C. Screening 25,000 transposon insertions revealed a total of 61 insertions that caused salt-sensitivity; and those insertions affected 31 genes. Only 12 of those genes were previously known to be required for salt-tolerance. Among the 61 insertions, three caused general osmo-sensitivity. We identified one single insertion mutant in the already-known gene, FPS1, required for survival of hypo-osmotic shock. A total of 31 insertions caused failure to grow at low temperature. Those identified ten different genes, three of which had previously been reported to affect cold-tolerance. Four genes were identified in both the salt and the cold-sensitivity screen. We found several unusual insertion mutations: (1) insertions in or close to essential genes, (2) insertion in an intergenic region and (3) insertions causing stress-sensitivity in W303-1A, while the deletion mutant in BY4741 did not show such a phenotype. Surprisingly, our mutant set and that reported in the large-scale transposon insertion project (TRIPLES, http://ygacmed.yale.edu/triples/triples.htm) only marginally overlap. We discuss some of the features of transposon mutagenesis in light of the availability of the complete set of yeast deletion mutants and we discuss the possible roles of the genes we identified.